Recent studies have implied that an additional transcription factor(s) beside the pituitary-specific factor Pit-1 is involved in homeostatic regulation of prolactin (PRL) promoter activity via Pit-1 binding cis active elements. We have employed one of these elements, site 1P, to clone from a pituitary cDNA expression library a protein, termed PBLF, that is a novel member of the WD repeat family. PBLF contains a PQ-rich potential transactivation domain, but no apparent DNA-binding motif. PBLF exhibits sequence-specific binding to site 1P, and is as active as Pit-1 in transactivating expression of PRL promoter constructs containing site 1P. In addition, PBLF, but not Pit-1, can mediate the transcriptional action of protein kinase A. In mouse embryo on day E12.5, PBLF is expressed specifically in Rathke's pouch, suggesting a role in pituitary development. PBLF accumulates in the nuclei of GH3 rat pituitary cells, and its mRNA is present in these cells at levels comparable to Pit-1 mRNA. PBLF transcripts are expressed in all adult human tissues examined. However, most tissues express multiple PBLF mRNA species that display tissue-specific patterns, suggesting that alternative splicing of the initial PBLF transcript may yield tissue- specific synthesis of isoforms of PBLF. These observations imply that PBLF will prove to play a significant transcriptional regulatory role, in the pituitary as well as in other organs; and thus provide the conceptual basis for our further investigations of the biochemical and functional properties of this novel protein. Biochemical studies will involve first production of soluble recombinant PBLF, followed by determination of the PBLF domain structure, characterization of its DNA- binding properties, and studies of the binding of PBLF to site 1P in the presence of Pit-1. Functional studies will involve investigations of the role of PBLF as a transcription factor in pituitary cells; of alternative splicing of the PBLF transcript in the brain; and of the PBLF phenotype, beginning with chromosomal localization of the PBLF gene in human, followed by attempts to identify a known organism (human or mouse) mutant for PBLF. These studies should provide further insight into the biochemical and physiological roles of PBLF.